British Columbia’s Experience after Implementation of the Treponema pallidum Reverse Algorithm and PCR Detection, 2015 to 2020

ABSTRACT British Columbia (BC) implemented the syphilis reverse screening algorithm and Treponema pallidum PCR testing in 2014. We summarize the performance characteristics of the algorithm, together with PCR direct detection, and report on syphilis cases identified from 2015 to 2020. Prior to 2015, samples for syphilis diagnosis were first screened by rapid plasma reagin (RPR). As of 2015, sera were screened by the Siemens Advia Centaur syphilis assay (enzyme immunoassay [EIA]). Positive and equivocal samples were reflex tested by a T. pallidum passive particle agglutination assay (TPPA) and RPR. We used T. pallidum DNA PCR on clinical samples and restriction fragment length polymorphism analysis to identify azithromycin resistance mutations. Case/epidemiological data were obtained from the BC surveillance system. Of 1,631,519 samples screened by the EIA, 72,492 (4.4%) were positive and 187 (<0.1%) were equivocal. Of EIA-positive/equivocal samples, 10.6% were false positive, and false positivity was higher at lower EIA indices. The reverse algorithm detected 4,693 late latent syphilis cases that likely would have been missed by RPR screening. PCR had a very high sensitivity of 100% versus 52.9% and 52.4% for dark-field (DF) and immunofluorescence (IF) microscopy, respectively. The azithromycin resistance mutation A2058G was identified in 96% of PCR-positive samples, and A2059G was identified in 4%. Annually, there were 944 to 1,467 syphilis cases, with 62% in men who reported male sexual partners. The reverse algorithm had a low false-positive rate and very few equivocal screening results but did identify previously undiagnosed late latent syphilis cases. PCR was more sensitive than both DF and IF microscopy for direct diagnosis and enabled monitoring for azithromycin resistance. IMPORTANCE In this study, we summarize the performance characteristics of the algorithm, together with PCR direct detection and epidemiological analysis, and report on syphilis cases identified from 2015 to 2020. This allowed us to paint a complete picture of the outcome of the utilization of the reverse algorithm for diagnosing syphilis cases. The study clearly showed that the reverse algorithm had a low false-positive rate and very few equivocal screening results but did identify previously undiagnosed late latent syphilis cases. PCR was more sensitive than both DF and IF microscopy for direct diagnosis and enabled monitoring for azithromycin resistance.

-The authors reference the BCCDC case definition website in the methods. However case definitions are often revised and optimized on a regular basis. For this reason the exact criteria used for syphilis staging should be detailed in the methods -On a similar theme the introduction could be better served by a brief statement describing the stages of syphilis infection.
-Provide primer sequences for the lab developed PCR test -The authors mention that penicillin resistance mutations have been identified but only conclude that azithromycin resistance should be monitored. Given that penicillin is the go to treatment , it is vital that surveillance of penicillin resistance also be highlighted given rising rates in Canada.
-Is prenatal screening for syphilis done in BC? If so what are the recommended screening intervals? This was not clear in the manuscript.
Reviewer #2 (Comments for the Author): Major Comments: A large retrospective study of the reverse algorithm and PCR-based Tp DNA detection from 2015-2020 was conducted in British Columbia. Azithromycin resistance in DNA positive samples was also examined during the same time period. Authors should explain the two different mutations and how they contribute to azithromycin resistance.
Lines 123-126: Since EIA is also better at detecting primary syphilis, a true syphilis case should also be determined by the following criteria: positive direct detection test (PCR, DF, or IF) and treponemal positivity (EIA and TPPA) or non-treponemal positivity (RPR). Line 174: Are these 4,693 EIA positives also TPPA positive? How did the authors determine these were latent cases vs primary vs previously treated or resolved? Were chart reviews conducted? How many were classified as early (confirmed/probable) vs late latent?
Line 178: Were these 814 samples tested by direct detection tests because they were primary cases from chancres? : How were congenital cases confirmed? How many congenital cases occurred prior to 2019?
Lines 201-204: With the rise of syphilis cases and the presumptive identification of latent cases, why do we not see more cases of neurosyphilis, ocular syphilis, and tertiary syphilis? My assumption is that most of these latent cases are previously treated or resolved infections. The authors mention that BC maintains a database of all syphilis diagnoses and treatments, but how many were unknowingly treated when taking antibiotics for other infections?
Lines 224-226: Authors should also mention that chancres may not be visible during primary infection if located inside the anal or vaginal cavity.
Lines 226-228: DF also requires a high bacterial burden (around 105 organisms/mL) unlike PCR which has an amplification step that increases sensitivity.
Line 234: The mainstay for syphilis treatment is penicillin. Alternative therapies include doxycycline, tetracycline, and ceftriaxone. Only in rare situations should azithromycin be used so it is unclear why this was included in the study.
Lines 250-252: What were the rates of congenital syphilis prior to 2019-2020?
Minor Comments: Lines 167-170: Might be good to mention again what is considered a false positive EIA+/TPPA-/RPR-. Also, would be nice to see number of false positives with percentages in parenthesis for 9.5% and 1.1%. Same for equivocal.

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Thank you for submitting your paper to Microbiology Spectrum. -On a similar theme the introduction could be better served by a brief statement describing the stages of syphilis infection.

Response to Reviewer Comments
We have included a description at the end of the first paragraph of the introduction.

-Provide primer sequences for the lab developed PCR test
We have included the primer sequences in the supplementary material.
-The authors mention that penicillin resistance mutations have been identified but only conclude that azithromycin resistance should be monitored. Given that penicillin is the go to treatment, it is vital that surveillance of penicillin resistance also be highlighted given rising rates in Canada.
We have added to the discussion a description of the reasons for monitoring azithromycin resistance and also commented on the importance of surveillance for penicillin resistance.
-Is prenatal screening for syphilis done in BC? If so what are the recommended screening intervals? This was not clear in the manuscript.
We have added to the discussion that prenatal screening is recommended but not mandatory in BC, and that the uptake exceeds 90%.
Reviewer #2 (Comments for the Author):

Major Comments:
A large retrospective study of the reverse algorithm and PCR-based Tp DNA detection from 2015-2020 was conducted in British Columbia. Azithromycin resistance in DNA positive samples was also examined during the same time period. Authors should explain the two different mutations and how they contribute to azithromycin resistance.
A2058G and A2059G are the Tp 23sRNA mutations that have been reported to be associated with macrolide treatment failures. It is unclear to us how their contributions to resistance are relevant to the present analysis. Therefore, we have not addressed this comment.
Lines 123-126: Since EIA is also better at detecting primary syphilis, a true syphilis case should also be determined by the following criteria: positive direct detection test (PCR, DF, or IF) and treponemal positivity (EIA and TPPA) or non-treponemal positivity (RPR).
We agree but the data were analyzed only on the basis of corresponding RPR results. We have included a statement in the limitations section that not including EIA positive results in this analysis may have resulted in lower calculated sensitivities for the direct tests. Line 174: Are these 4,693 EIA positives also TPPA positive? How did the authors determine these were latent cases vs primary vs previously treated or resolved? Were chart reviews conducted? How many were classified as early (confirmed/probable) vs late latent?
We have added a statement that these cases underwent chart review. We do not have information on whether they were classified as early vs. late latent; we only had information on whether they received syphilis treatment in the past.
Line 178: Were these 814 samples tested by direct detection tests because they were primary cases from chancres?
Yes, these were suspected syphilis lesions and this has been indicated in the manuscript.
Lines 194-195: How were congenital cases confirmed? How many congenital cases occurred prior to 2019? Table 3).

Congenital cases were confirmed by laboratory results and clinical assessment. There were no congenital cases identified for the years 2015-2018 (as shown in
Lines 201-204: With the rise of syphilis cases and the presumptive identification of latent cases, why do we not see more cases of neurosyphilis, ocular syphilis, and tertiary syphilis?
My assumption is that most of these latent cases are previously treated or resolved infections. The authors mention that BC maintains a database of all syphilis diagnoses and treatments, but how many were unknowingly treated when taking antibiotics for other infections?
We agree that both spontaneous resolution and unknowing treatment are the most likely explanations and we have updated the discussion accordingly. We have no data on how many individuals might have received antibiotic treatment for other indications.
Lines 224-226: Authors should also mention that chancres may not be visible during primary infection if located inside the anal or vaginal cavity.

We agree and have added this point to the discussion.
Lines 226-228: DF also requires a high bacterial burden (around 105 organisms/mL) unlike PCR which has an amplification step that increases sensitivity.
While this seems self-evident, we have nevertheless added a statement to the discussion.
Line 234: The mainstay for syphilis treatment is penicillin. Alternative therapies include doxycycline, tetracycline, and ceftriaxone. Only in rare situations should azithromycin be used so it is unclear why this was included in the study.
We have added a statement to the discussion stating the reason for monitoring azithromycin resistance.
Lines 250-252: What were the rates of congenital syphilis prior to 2019-2020? Table 3 shows that no cases of congenital syphilis were diagnosed from 2015-2018.

Minor Comments:
Lines 167-170: Might be good to mention again what is considered a false positive EIA+/TPPA-/RPR-. Also, would be nice to see number of false positives with percentages in parenthesis for 9.5% and 1.1%. Same for equivocal.
We have added this information. There were some errors in the original numbers cited and these have been corrected. It is my pleasure to tell you that your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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